Temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal communities with ontogenetic and phenological development in Prasophyllum (Orchidaceae)

Abstract

Background and aims: Plant-fungus symbioses may experience temporal turnover during the ontogenetic or phenological development of the host, which can influence the ecological requirements of the host plant. In this study, we investigate temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal (OMF) communities in Prasophyllum (Orchidaceae), asking whether OMF communities are subject to temporal change attributable to orchid phenology or ontogeny.

Methods: Roots of adult Prasophyllum frenchii, Prasophyllum lindleyanum and Prasophyllum sp. aff. validum from Australia were sampled between autumn and spring. Seed was sown in situ as 'baits' to explore the mycorrhizal associations of germinating protocorms, which were compared with OMF in roots of co-occurring adult plants. Culture-dependent and -independent sequencing methods were used to amplify the internal transcribed spacer and mitochondrial large subunit loci, with sequences assigned to operational taxonomic units (OTUs) in phylogenetic analyses. Germination trials were used to determine whether fungal OTUs were mycorrhizal.

Key results: A persistent core of OMF was associated with Prasophyllum, with Ceratobasidiaceae OMF dominant in all three species. Phenological turnover occurred in P. lindleyanum and P. sp. aff. validum, but not in P. frenchii, which displayed specificity to a single OTU. Ontogenetic turnover occurred in all species. However, phenological and ontogenetic turnover was typically driven by the presence or absence of infrequently detected OTUs in populations that otherwise displayed specificity to one or two dominant OTUs. Ex situ germination trials showed that 13 of 14 tested OTUs supported seed germination in their host orchid, including eight OTUs that were not found in protocorms in situ.

Conclusions: An understanding of OMF turnover can have practical importance for the conservation of threatened orchids and their mycorrhizal partners. However, frameworks for classifying OMF turnover should focus on OTUs important to the life cycle of the host plant, which we suggest are likely to be those that are frequently detected or functionally significant.

Keywords: Prasophyllum; Ceratobasidiaceae; Orchidaceae; conservation; fungi; mycorrhiza; ontogenetic; orchid; phenology; seed baiting; temporal turnover.

Fig. 1.

Study species, from left: Prasophyllum frenchii, Prasophyllum lindleyanum and Prasophyllum sp. aff. validum.

Fig. 2.

Map of study sites in Victoria, Australia, and charts of mean monthly rainfall (in millimetres). Data are from the nearest currently operating weather stations: Stawell (data from 1996–2020), Warby Ranges (2002–2020) and Yarram (2007–2020) (BOM, 2021).

Fig. 3.

Maps of study sites of Prasophyllum frenchii (A), Prasophyllum lindleyanum (B) and Prasophyllum sp. aff. validum (C), showing the spatial location of sampled adult plants and protocorms, the operational taxonomic units (OTUs) with which they associated, and (for adult plants) the season of sampling. Circles representing adult plants are split according to the number and identity of OTUs recorded per plant (not relative frequency of detection).

Fig. 4.

Relative frequency of operational taxonomic units (OTUs; as a percentage of all sequences) in roots of adult plants of Prasophyllum frenchii (205 sequences), Prasophyllum lindleyanum (254 sequences) and Prasophyllum sp. aff. validum (235 sequences) at different sampling intervals throughout the 2018 season from autumn to spring, and in protocorms in spring 2018 (P. frenchii = 86 sequences, P. lindleyanum = 146 sequences and P. sp. aff. validum = 54 sequences).

Fig. 5.

Maximum likelihood phylogeny of concatenated ITS and mitochondrial large subunit (mtLSU) sequences of all orchid mycorrhizal fungi associated with protocorms and adult plants in this study across Prasophyllum frenchii, Prasophyllum lindleyanum and Prasophyllum sp. aff. validum. Branch labels are bootstrap values (>70 shown); labels on minor branches are not shown.

Fig. 6.

Pairwise comparisons of β_total against β_rich for sampling periods measuring seasonal (grey) and ontogenetic (green) turnover in Prasophyllum frenchii (squares), Prasophyllum lindleyanum (circles) and Prasophyllum sp. aff. validum (triangles). Negative and positive change in operational taxonomic unit (OTU) richness indicates a decrease or increase in the number of OTUs from protocorm to adult life stages or from earlier to later seasonal sampling periods. Comparisons are grouped and labelled according to the turnover scenarios of Ventre Lespiaucq et al. (2021): specificity to a single OTU (a1) and to multiple OTUs (a4); nested OTU gain (b2) and loss (b3); and partial OTU replacement with loss (c3) and partial replacement with constant richness (c4).